Emergence of mcr-3 carrying Escherichia coli in Diseased Pigs in South Korea

We examined the prevalence and molecular characteristics of mcr-3 carrying colistin-resistant Escherichia coli among cattle, pig, and chicken isolates in South Korea. Among a total of 185 colistin-resistant E. coli isolates determined in this study (47 from cattle, 90 from pigs, and 48 from chicken), PCR amplification detected mcr-3 genes in 17 isolates predominantly from diseased pigs. The mcr-3 genes were characterized as mcr-3.1 in 15 isolates and mcr-3.5 in 2 isolates. The mcr-3 gene was transferred to the E. coli J53 recipient strain from more than 50% of the mcr-3-carrying isolates. The mcr-3.1 and mcr-3.5 genes were identified predominantly in IncHI2 and IncP plasmids, respectively. Multi-locus sequence typing analysis revealed eight previously reported sequence types (ST), including ST1, ST10, and ST42. We identified isolates with similar pulsed-field gel electrophoresis patterns from diseased pigs in three farms. Besides, the isolates carried various virulence factors and demonstrated resistance to multiple antimicrobials, including β-lactams and quinolones. Further, the mcr-3.5 encodes three amino acid substitutions compared with mcr-3.1. To the best of our knowledge, this is the first report of pathogenic E. coli carrying mcr-3.5 in South Korea, which implies that mcr-3 variants may have already been widely spread in the pig industry.

Detection of mcr-1 Gene among Escherichia coli Isolates from Farmed Fish and Characterization of mcr-1 -Bearing IncP Plasmids

ABSTRACT The presence of the mcr-1 gene in Escherichia coli isolated from retail freshwater fish was investigated. Seven (3.65%) clonally unrelated original E. coli isolates from grass carp were positive for mcr-1 . The mcr-1 genes were encoded by either chromosomes ( n = 2) or conjugative plasmids (2 IncI2, 2 IncP, and 1 IncX4). The IncP plasmids were similar to other mcr-1 -harboring IncP plasmids from China, though the insertion sites varied. Our report warrants further surveillance of resistance genes in aquaculture.

Characterization of a P1-like bacteriophage carrying CTX-M-27 in Salmonella spp. resistant to third generation cephalosporins isolated from pork in China

The aim of this study was to elucidate the epidemiology of third generation cephalosporin resistant Samonella isolates from pork of a slaughterhouse in China and the features of transferable elements carrying bla CTX-M genes. One hundred and twenty-six (7.3%) Salmonella isolates were identified; S. Derby and S. Rissen were the most two prevalent serotypes. Among these isolates 20 (15.8%) were resistant to third generation cephalosporins and nine of them carried bla CTX-M-27. S1-PFGE and replicon typing of bla CTX-M-27-carrying plasmids showed that seven were untypeable plasmids of about 104 Kb and two were IncP plasmids of about 300 Kb. Complete sequence analysis of one PBRT-untypeable plasmid showed it was a P1-like bateriophage, named SJ46, which contained a non-phage-associated region with several mobile elements, including Tn1721, ISEcp1B and IS903D. The other six 104 Kb PBRT-untypeable bla CTX-M-27-carrying plasmids also harboured the same phage-insertion region of SJ46 suggesting that they were the same P1-like bacteriophage. PFGE profiles of the parental strains revealed both potential vertical and horizontal spread of this P1-like bla CTX-M-27-containing element. Additionally, the representative gene of the P1 family bacteriophage, repL, was detected in 19.0% (24/126) of the isolates. This study indicated a potential role of P1-family bacteriophage in capture and spread of antimicrobial resistance in pathogens.

Embedded elements in the IncPβ plasmids R772 and R906 can be mobilized and can serve as a source of diverse and novel elements

IncP plasmids are important contributors to bacterial adaptation. Their phenotypic diversity is due largely to accessory regions located in one or two specific parts of the plasmid. The accessory regions are themselves diverse, as judged from sequenced plasmids mostly isolated from non-clinical sources. To further understand the diversity, evolutionary history and functional attributes of the accessory regions, we compared R906 and R772, focusing on the oriV–trfA accessory region. These IncPβ plasmids were from porcine and clinical sources, respectively. We found that the accessory regions formed potentially mobile elements, Tn510 (from R906) and Tn511 (from R772), that differed internally but had identical borders. Both elements appeared to have evolved from a TnAO22-like mer transposon that had inserted into an ancestral IncPβ plasmid and then accrued additional transposable elements and genes from various proteobacteria. Structural comparisons suggested that Tn510 (and a descendent in pB10), Tn511 and the mer element in pJP4 represent three lineages that evolved from the same widely dispersed IncPβ carrier. Functional studies on Tn511 revealed that its mer module is inactive due to a merT mutation, and that its aphAI region is prone to deletion. More significantly, we showed that by providing a suitable transposase gene in trans, the defective Tn510 and Tn511 could transpose intact or in part, and could also generate new elements (stable cointegrates and novel transposons). The ingredients for assisted transposition events similar to those observed here occur in natural microcosms, providing non-self-mobile elements with avenues for dispersal to new replicons and for structural diversification. This work provides an experimental demonstration of how the complex embedded elements uncovered in IncP plasmids and in other plasmid families may have been generated.

Predicting Plasmid Promiscuity Based on Genomic Signature

ABSTRACT Despite the important contribution of self-transmissible plasmids to bacterial evolution, little is understood about the range of hosts in which these plasmids have evolved. Our goal was to infer this so-called evolutionary host range. The nucleotide composition, or genomic signature, of plasmids is often similar to that of the chromosome of their current host, suggesting that plasmids acquire their hosts’ signature over time. Therefore, we examined whether the evolutionary host range of plasmids could be inferred by comparing their trinucleotide composition to that of all completely sequenced bacterial chromosomes. The diversity of candidate hosts was determined using taxonomic classification and genetic distance. The method was first tested using plasmids from six incompatibility (Inc) groups whose host ranges are generally thought to be narrow (IncF, IncH, and IncI) or broad (IncN, IncP, and IncW) and then applied to other plasmid groups. The evolutionary host range was found to be broad for IncP plasmids, narrow for IncF and IncI plasmids, and intermediate for IncH and IncN plasmids, which corresponds with their known host range. The IncW plasmids as well as several plasmids from the IncA/C, IncP, IncQ, IncU, and PromA groups have signatures that were not similar to any of the chromosomal signatures, raising the hypothesis that these plasmids have not been ameliorated in any host due to their promiscuous nature. The inferred evolutionary host range of IncA/C, IncP-9, and IncL/M plasmids requires further investigation. In this era of high-throughput sequencing, this genomic signature method is a useful tool for predicting the host range of novel mobile elements.

Conjugative Type 4 Secretion System of a Novel Large Plasmid from the Chemoautotroph Tetrathiobacter kashmirensis and Construction of Shuttle Vectors for Alcaligenaceae

ABSTRACT Tetrathiobacter spp. and other members of the Alcaligenaceae are metabolically versatile and environmentally significant. A novel, ∼60-kb conjugative plasmid, pBTK445, from the sulfur chemolithoautotroph Tetrathiobacter kashmirensis, was identified and characterized. This plasmid exists at a low copy number of 2 to 3 per host chromosome. The portion of pBTK445 sequenced so far (∼25 kb) harbors genes putatively involved in replication, transfer functions, partition, and UV damage repair. A 1,373-bp region was identified as the minimal replicon. This region contains a repA gene encoding a protein belonging to the RPA (replication protein A) superfamily and an upstream, iteron-based oriV. A contiguous 11-gene cluster homologous to various type 4 secretion systems (T4SSs) was identified. Insertional inactivation demonstrated that this cluster is involved in the conjugative transfer functions of pBTK445, and thus, it was named the tagB (transfer-associated gene homologous to virB) locus. The core and peripheral TagB components show different phylogenetic affinities, suggesting that this system has evolved by assembling components from evolutionarily divergent T4SSs. A virD4 homolog, putatively involved in nucleoprotein transfer, is also present downstream of the tagB locus. Although pBTK445 resembles IncP plasmids in terms of its genomic organization and the presence of an IncP-specific trbM homolog, it also shows several unique features. Unlike that of IncP, the oriT of pBTK445 is located in close proximity to the oriV, and a traL homolog, which is generally present in the TraI locus of IncP, is present in pBTK445 in isolation, upstream of the tagB locus. A significant outcome of this study is the construction of conjugative shuttle vectors for Tetrathiobacter and related members of the Alkaligenaceae.

Characterization of the Replication, Maintenance, and Transfer Features of the IncP-7 Plasmid pCAR1, Which Carries Genes Involved in Carbazole and Dioxin Degradation

ABSTRACT Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 � 10−1 and 3 � 10−3 per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.

Analysis of the Mobilization Region of the Broad-Host-Range IncQ-Like Plasmid pTC-F14 and Its Ability To Interact with a Related Plasmid, pTF-FC2

ABSTRACT Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.